Modulators of β-amyloid peptide aggregation

ABSTRACT

Compounds that act to modulate the aggregation of natural β amyloid peptides (β-AP) are disclosed. The β amyloid modulators of the invention can promote β-AP aggregation or, more preferably, can inhibit natural β-AP aggregation. Furthermore, the modulators are capable of altering natural β-AP aggregation when the natural β-APs are in a molar excess amount relative to the modulators. Pharmaceutical compositions comprising the compounds of the invention, and methods of altering natural β-AP aggregation using the compounds of the invention, are also disclosed.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/404,831, filed Mar. 14, 1995, pending, the entire contentsof which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD), first described by the Bavarian psychiatristAlois Alzheimer in 1907, is a progressive neurological disorder thatbegins with short term memory loss and proceeds to disorientation,impairment of judgement and reasoning and, ultimately, dementia. Thecourse of the disease usually leads to death in a severely debilitated,immobile state between four and 12 years after onset. AD has beenestimated to afflict 5 to 11 percent of the population over age 65 andas much as 47 percent of the population over age 85. The societal costfor managing AD is upwards of 80 billion dollars annually, primarily dueto the extensive custodial care required for AD patients. Moreover, asadults born during the population boom of the 1940's and 1950's approachthe age when AD becomes more prevalent, the control and treatment of ADwill become an even more significant health care problem. Currently,there is no treatment that significantly retards the progression of thedisease. For reviews on AD, see Selkoe, D. J. Sci. Amer., November 1991,pp. 68-78; and Yankner, B. A. et al. (1991) N. Eng. J. Med.325:1849-1857.

It has recently been reported (Games et al. (1995) Nature 373:523-527)that an Alzheimer-type neuropathology has been created in transgenicmice. The transgenic mice express high levels of human mutant amyloidprecurser protein and progressively develop many of the pathologicalconditions associated with AD.

Pathologically, AD is characterized by the presence of distinctivelesions in the victim's brain. These brain lesions include abnormalintracellular filaments called neurofibrillary tangles (NTFs) andextracellular deposits of amyloidogenic proteins in senile, or amyloid,plaques. Amyloid deposits are also present in the walls of cerebralblood vessels of AD patients. The major protein constituent of amyloidplaques has been identified as a 4 kilodalton peptide called β-amyloidpeptide (β-AP)(Glenner, G. G. and Wong, C. W. (1984) Biochem. Biophys.Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl. Acad.Sci. USA 82:4245-4249). Diffuse deposits of β-AP are frequently observedin normal adult brains, whereas AD brain tissue is characterized by morecompacted, dense-core β-amyloid plaques. (See e.g., Davies, L. et al.(1988) Neurology 38:1688-1693). These observations suggest that β-APdeposition precedes, and contributes to, the destruction of neurons thatoccurs in AD. In further support of a direct pathogenic role for β-AP,β-amyloid has been shown to be toxic to mature neurons, both in cultureand in vivo. Yankner, B. A. et al. (1989) Science 245:417-420; Yankner,B. A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9020-9023; Roher, A.E. et al. (1991) Biochem. Biophys. Res. Commun. 174:572-579; Kowall, N.W. et al. (1991) Proc. Natl. Acad. Sci. USA 88:7247-7251. Furthermore,patients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type(HCHWA-D), which is characterized by diffuse β-amyloid deposits withinthe cerebral cortex and cerebrovasculature, have been shown to have apoint mutation that leads to an amino acid substitution within β-AP.Levy, E. et al. (1990) Science 248:1124-1126. This observationdemonstrates that a specific alteration of the β-AP sequence can causeβ-amyloid to be deposited.

Natural β-AP is derived by proteolysis from a much larger protein calledthe amyloid precursor protein (APP). Kang, J. et al. (1987) Nature325:733; Goldgaber, D. et al. (1987) Science 235:877; Robakis, N. K. etal. (1987) Proc. Natl. Acad. Sci. USA 84:4190; Tanzi, R. E. et al.(1987) Science 235:880. The APP gene maps to chromosome 21, therebyproviding an explanation for the β-amyloid deposition seen at an earlyage in individuals with Down's syndrome, which is caused by trisomy ofchromosome 21. Mann, D. M. et al. (1989) Neuropathol. Appl. Neurobiol.15:317; Rumble, B. et al. (1989) N. Eng. J. Med. 320:1446. APP containsa single membrane spanning domain, with a long amino terminal region(about two-thirds of the protein) extending into the extracellularenvironment and a shorter carboxy-terminal region projecting into thecytoplasm. Differential splicing of the APP messenger RNA leads to atleast five forms of APP, composed of either 563 amino acids (APP-563),695 amino acids (APP-695), 714 amino acids (APP-714), 751 amino acids(APP-751) or 770 amino acids (APP-770).

Within APP, naturally-occurring β amyloid peptide begins at an asparticacid residue at amino acid position 672 of APP-770. Naturally-occurringβ-AP derived from proteolysis of APP is 39 to 43 amino acid residues inlength, depending on the carboxy-terminal end point, which exhibitsheterogeneity. The predominant circulating form of β-AP in the blood andcerebrospinal fluid of both AD patients and normal adults is β1-40("short β"). Seubert, P. et al. (1992) Nature 359:325; Shoji, M. et al.(1992) Science 258:126. However, β1-42 and β1-43 ("long β") also areforms in β-amyloid plaques. Masters, C. et al. (1985) Proc. Natl. Acad.Sci. USA 82:4245; Miller, D. et al. (1993) Arch. Biochem. Biophys.301:41; Mori, H. et al. (1992) J. Biol. Chem. 267:17082. Although theprecise molecular mechanism leading to β-APP aggregation and depositionis unknown, the process has been likened to that of nucleation-dependentpolymerizations, such as protein crystallization, microtubule formationand actin polymerization. See e.g., Jarrett, J. T. and Lansbury, P. T.(1993) Cell 73:1055-1058. In such processes, polymerization of monomercomponents does not occur until nucleus formation. Thus, these processesare characterized by a lag time before aggregation occurs, followed byrapid polymerization after nucleation. Nucleation can be accelerated bythe addition of a "seed" or preformed nucleus, which results in rapidpolymerization. The long β forms of β-AP have been shown to act asseeds, thereby accelerating polymerization of both long and short β-APforms. Jarrett, J. T. et al. (1993) Biochemistry 32:4693.

In one study, in which amino acid substitutions were made in β-AP, twomutant β peptides were reported to interfere with polymerization ofnon-mutated β-AP when the mutant and non-mutant forms of peptide weremixed. Hilbich, C. et al. (1992) J. Mol. Biol. 228:460-473. However,equimolar amounts of the mutant and non-mutant (i.e., natural) β amyloidpeptides were used to see this effect and the mutant peptides werereported to be unsuitable for use in vivo. Hilbich, C. et al. (1992),supra.

SUMMARY OF THE INVENTION

The present invention provides β amyloid modulators that affect theaggregation of natural β-amyloid and therefore can be used to treatsubjects having a disorder associated with β-amyloidosis, e.g.Alzheimer's disease (AD). The aggregation and deposition of β-amyloidplay an important role in the pathology of AD. The modulators of thepresent invention can affect aggregation of natural β amyloid peptideswhen present at a lower concentration than that of the natural β amyloidpeptides and further are suitable for therapeutic use in vivo.

This invention generally pertains to compounds that are capable ofaltering the aggregation of natural β amyloid peptides (β-AP) when thecompounds of the invention are contacted with the natural β-AP. Thesecompounds, referred to as β amyloid modulators, either can promote β-APaggregation or, more preferably, can inhibit β-AP aggregation. Moreover,the preferred modulators of the invention are capable of alteringnatural β-AP aggregation even when the natural β-AP concentration is inmolar excess compared to the concentration of the modulator. Thus, asmall amount of a modulator of the invention acts to disrupt the naturalβ amyloid aggregation process or rate. Because of their ability toalter, and preferably inhibit, natural β-AP aggregation, the modulatorsof the invention are useful therapeutically in the treatment ofdisorders associated with β amyloidosis, in particular Alzheimer'sdisease. Moreover, the modulators of the invention can be useddiagnostically in assays to detect and quantitate natural β-AP in an invitro sample, such as a sample of biological fluid.

In one embodiment, a modulator of the invention is a β-amyloid peptidecompound comprising a formula: ##STR1## wherein Xaa is a β-amyloidpeptide, A is a modulating group attached directly or indirectly to theβ-amyloid peptide of the compound such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides, and n is an integer selected such that thecompound inhibits aggregation of natural β-amyloid peptides whencontacted with the natural β-amyloid peptides.

Preferably, the β-amyloid peptide of the compound has an amino-terminalamino acid residue corresponding to position 668 of β-amyloid precursorprotein-770 (APP-770), or to a residue carboxy-terminal to position 668.Even more preferably, the β-amyloid peptide of the compound has anamino-terminal amino acid residue corresponding to position 672 ofAPP-770, or to a residue carboxy-terminal to position 672. Preferably,the β-amyloid peptide of the compound has between 6 and 60 amino acidresidues, more preferably between 10 and 43 amino acid residues, andeven more preferably between 10 and 25 amino acid residues. A preferredβ-amyloid peptide of the compound consists of an amino acid sequenceshown in SEQ ID NO: 2, or an amino-terminal or carboxy-terminal deletionthereof having at least 6 amino acid residues.

A modulating group(s) ("A") can be attached to the β-amyloid peptide ofthe compound through, for example, the amino terminus of the peptide,the carboxy-terminus of the peptide or the side chain(s) of one or moreamino acid residues. The number of modulating groups ("n") attached tothe peptide is selected such that the β-amyloid peptide is capable ofperforming its intended function of modulating aggregation of naturalβ-amyloid peptides. The number of modulating groups is preferablybetween 1 and 60, more preferably between 1 and 30 and even morepreferably between 1 and 10 or 1 and 5. A preferred modulating group, inparticular for modifying the amino terminus of the β amyloid peptide ofthe compound, is a biotin compound.

In another embodiment, the invention provides a modulator of β-amyloidaggregation which alters aggregation of natural β-amyloid peptides whencontacted with a molar excess amount of natural β-amyloid peptides. Themodulator may promote or inhibit aggregation of natural β-amyloidpeptides, and preferably alters β-AP aggregation of at least a 10-foldmolar excess amount of natural β-AP. More preferably, the modulatoralters aggregation of at least a 100-fold molar excess amount of naturalβ-AP.

Methods for selecting a modulator of β-amyloid aggregation of theinvention are also provided. In a preferred embodiment, the methodinvolves contacting a test compound with an molar excess amount ofnatural β-amyloid peptides (β-AP), measuring the aggregation of thenatural β-AP in the presence of the test compound and selecting a testcompound that reduces the aggregation of the natural β-AP, compared tothe aggregation of the natural β-AP in the absence of the test compound.Aggregation of the natural β-AP can be measured, for example bymeasuring turbidity of a solution of the natural β-AP in the presence ofthe test compound.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising a modulator of the invention in a therapeutically effectiveamount sufficient to alter, and preferably inhibit, aggregation ofnatural β-amyloid peptides, and a pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier can be suitable for parenteraladministration. The carrier also can be suitable for intrathecaladministration (e.g., intraspinal or intracerebral administration). Thepharmaceutical composition of the invention can be provided as apackaged formulation including, for example, the composition in acontainer and instructions for administration of the composition.

Yet another aspect of the invention pertains to a method for altering,and preferably inhibiting, aggregation of natural β-amyloid peptides.The method involves contacting the natural β-amyloid peptides with amodulator of the invention such that aggregation of the naturalβ-amyloid peptides is altered, and preferably inhibited. In oneembodiment of the method, the modulator is contacted with a molar excessamount of natural β-AP. The method of the invention can be used todetect and quantitate natural β-AP in vitro (e.g., in a biologicalsample). Furthermore, a modulator can be contacted with natural β-AP ina subject (e.g., in the brain parenchyma or cerebrospinal fluid of thesubject) by administering the modulator to the subject to thereby alter,and preferably inhibit, natural β-AP aggregation in the subject. Themodulator preferably is administered directly into the central nervoussystem (CNS) of the subject. For example, the modulator may beadministered into the CNS through a catheter (e.g., intraspinally), viaa surgically implanted infusion pump, through a reservoir (e.g.,intracerebrally) or by direct injection; (e.g., lumbar puncture).Accordingly, medical devices such as syringes, pumps, reservoirs,catheters and the like containing a pharmaceutical composition of theinvention are also encompassed by the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graphic representation of the turbidity of a β-AP₁₋₄₀solution, as measured by optical density at 400 nm, either in theabsence of a β-amyloid modulator or in the presence of the β-amyloidmodulator N-biotinyl-βAP₁₋₄₀ (1%, or 5%).

FIG. 2 is a schematic representation of compounds which can be used tomodify a β-AP to form a β-amyloid modulator of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to compounds, and pharmaceutical compositionsthereof, that can modulate the aggregation of natural β amyloid peptides(β-AP). A compound of the invention, referred to herein interchangeablyas a β amyloid peptide modulator, a β amyloid modulator or simply amodulator, alters the aggregation of natural β-AP when the modulator iscontacted with natural β-AP. Thus, a compound of the invention acts toalter the natural aggregation process or rate for β-AP, therebydisrupting this process.

Accordingly, as used herein, a "modulators" of β-amyloid aggregation isintended to refer to an agent that, when contacted with natural βamyloid peptides, alters the aggregation of the natural β amyloidpeptides. The term "aggregation of β amyloid peptides" refers to aprocess whereby the peptides associate with each other to form amultimeric, largely insoluble complex. The term "aggregation" further isintended to encompass β amyloid fibril formation and also encompassesβ-amyloid plaques.

The terms "natural β-amyloid peptide" and "natural β-AP", usedinterchangeably herein, are intended to encompass naturally occurringproteolytic cleavage products of the β amyloid precursor protein (APP),including β amyloid peptides having 39-43 amino acids (i.e., β1-39,β1-40, β1-41, β1-42, β1-43). The amino-terminal acid residue of naturalβ-AP corresponds to the aspartic acid residue at position 672 of the 770amino acid residue form of APP ("APP-770"). The 43 amino acid long formof natural β-AP has the amino acid sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

(SEQ ID NO: 1), whereas the shorter forms have 1-4 amino acid residuesdeleted from the carboxy-terminal end.

In the presence of a modulator of the invention, aggregation of naturalβ amyloid peptides is "altered". The various forms of the term"alteration" are intended to encompass both inhibition of β-APaggregation and promotion of β-AP aggregation. Aggregation of naturalβ-AP is "inhibited" in the presence of the modulator when there is adecrease in the amount and/or rate of β-AP aggregation as compared tothe amount and/or rate of β-AP aggregation in the absence of themodulator. The various forms of the term "inhibition" are intended toinclude both complete and partial inhibition of β-AP aggregation.Preferably, aggregation is inhibited at least 10%, more preferably, atleast 20%, 30%, 40% or 50%. Alternatively, the various forms of the term"promotion" refer to an increase in the amount and/or rate of β-APaggregation in the presence of the modulator, as compared to the amountand/or rate of β-AP aggregation in the absence of the modulator.

In a preferred embodiment, the modulators of the invention are capableof altering β-AP aggregation when contacted with a molar excess amountof natural β-AP. A "molar excess amount of natural β-AP" refers to aconcentration of natural β-AP, in moles, that is greater than theconcentration, in moles, of the modulator. For example, if the modulatorand β-AP are both present at a concentration of 1 μM, they are said tobe "equimolar", whereas if the modulator is present at a concentrationof 1 μM and the β-AP is present at a concentration of 5 μM, the β-AP issaid to be present at a 5-fold molar excess amount compared to themodulator. Preferably, a modulator of the invention is effective ataltering natural β-AP aggregation when the natural β-AP is present at atleast a 5-fold, and more preferably at least a 10-fold, 20-fold,50-fold, 100-fold, 500-fold or 1000-fold molar excess compared to theconcentration of the modulator.

In one embodiment, a modulator of the invention is a β-amyloid peptidecompound comprising the formula: ##STR2##

wherein Xaa is a β-amyloid peptide, A is a modulating group attacheddirectly or indirectly to the β-amyloid peptide of the compound suchthat the compound inhibits aggregation of natural β-amyloid peptideswhen contacted with the natural β-amyloid peptides, and n is an integerselected such that the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.

Preferably, β-amyloid peptide of the compound has an amino-terminalamino acid residue corresponding to position 668 of β-amyloid precursorprotein-770 (APP-770) or to a residue carboxy-terminal to position 668of APP-770. The amino acid sequence of APP-770 from position 668 toposition 770 (i.e., the carboxy terminus) is shown below and in SEQ IDNO: 2:

EVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITL

VMLKKKQYTSIHHGVVEVDAAVTPEERHLSKMQQNGYENPTYKFFEQMQN.

More preferably, the amino-terminal amino acid residue of the β-amyloidpeptide corresponds to position 672 of APP-770 (position 5 of the aminoacid sequence of SEQ ID NO: 2) or to a residue carboxy-terminal toposition 672 of APP-770. Although the β-amyloid peptide of the compoundmay encompass the 103 amino acid residues corresponding to positions668-770 of APP-770, preferably the peptide is between 6 and 60 aminoacids in length, more preferably between 10 and 43 amino acids in lengthand even more preferably between 10 and 25 amino acid residues inlength.

As used herein, the term "β amyloid peptide", as used in a modulator ofthe invention is intended to encompass peptides having an amino acidsequence identical to that of the natural sequence in APP, as well aspeptides having acceptable amino acid substitutions from the naturalsequence. Acceptable amino acid substitutions are those that do notaffect the ability of the peptide to alter natural β-AP aggregation.Moreover, particular amino acid substitutions may further contribute tothe ability of the peptide to alter natural β-AP aggregation and/or mayconfer additional beneficial properties on the peptide (e.g., increasedsolubility, reduced associated with other amyloid proteins, etc). Forexample, substitution of hydrophobic amino acid residues for the twophenylalanine residues at positions 19 and 20 of natural β-AP (positions19 and 20 of the amino acid sequence shown in SEQ ID NO: 1) may furthercontribute to the ability of the peptide to alter β-AP aggregation (seeHilbich, C. (1992) J. Mol. Biol. 228:460-473). Thus, in one embodiment,the β-AP of the compound consists of the amino acid sequence shown belowand in SEQ ID NO: 3:

DAEFRHDSGYEVHHQKLV(Xaa₁₉)(Xaa₂₀)AEDVGSNKGAIIGLMVGGVVIAT

(or an amino-terminal or carboxy-terminal deletion thereof), wherein Xaais a hydrophobic amino acid. Examples of hydrophobic amino acids areisoleucine, leucine, threonine, serine, alanine, valine or glycine.Preferably, F₁₉ F₂₀ is substituted with T₁₉ T₂₀ or G₁₉ I₂₀.

Other suitable amino acid substitutions include replacement of aminoacids in the human peptide with the corresponding amino acids of therodent β-AP peptide. The three amino acid residues that differ betweenhuman and rat β-AP are at positions 5, 10 and 13 of the amino acidsequence shown in SEQ ID NOs: 1 and 3. A human β-AP having the human torodent substitutions Arg₅ to Gly, Tyr₁₀ to Phe and His₁₃ to Arg has beenshown to retain the properties of the human peptide (see Fraser, P. E.et al. (1992) Biochemistry 31:10716-10723; and Hilbich, C. et al. (1991)Eur. J. Biochem. 201:61-69). Accordingly, a human β-AP having rodentβ-AP a.a. substitutions is suitable for use in a modulator of theinvention.

Other possible β-AP amino acid substitutions are described in Hilbich,C. et al. (1991) J. Mol. Biol. 218:149-163; and Hilbich, C. (1992) J.Mol. Biol. 228:460-473. Moreover, amino acid substitutions that affectthe ability of β-AP to associate with other proteins can be introduced.For example, one or more amino acid substitutions that reduce theability of β-AP to associate with the serpin enzyme complex (SEC)receptor, α1-antichymotrypsin (ACT) and/or apolipoprotein E (ApoE) canbe introduced. A preferred substitution for reducing binding to the SECreceptor is L₃₄ M₃₅ to A₃₄ A₃₅ (at positions 34 and 35 of the amino acidsequences shown in SEQ ID NOs: 1 and 3). A preferred substitution forreducing binding to ACT is S₈ to A₈ (at position 8 of the amino acidsequences shown in SEQ ID NOs: 1 and 3).

Alternative to β-AP amino acid substitutions described herein or knownin the art, a modulator composed, at least in part, of an aminoacid-substituted β amyloid peptide can be prepared by standardtechniques and tested for the ability to alter β-AP aggregation using anaggregation assay described herein. To retain the properties of theoriginal modulator, preferably conservative amino acid substitutions aremade at one or more amino acid residues, A "conservative amino acidsubstitution" is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),β-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Accordingly, a modulator composed of a β amyloid peptidehaving an amino acid sequence that is mutated from that of the wild-typesequence in APP-770 yet which still retains the ability to alter naturalβ-AP aggregation is within the scope of the invention.

As used herein, the term "β amyloid peptide" is further intended toinclude peptide analogues or peptide derivatives or peptidomimetics thatretain the ability to alter natural β-AP aggregation as describedherein. For example, a β amyloid peptide of a modulator of the inventionmay be modified to increase its stability, bioavailability, solubility,etc. The terms "peptide analogue", "peptide derivative" and"peptidomimetic" as used herein are intended to include molecules whichmimic the chemical structure of a peptide and retain the functionalproperties of the peptide. Approaches to designing peptide analogs areknown in the art. For example, see Farmer, P. S. in Drug Design (E. J.Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball.J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A.and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues,derivatives and peptidomimetics include peptides substituted with one ormore benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942), peptides with methylated amide linkages and"retro-inverso" peptides (see U.S. Pat. No. 4,522,752 by Sisto).

In a modulator of the invention having the formula shown above, amodulating group ("A") is attached directly or indirectly to theβ-amyloid peptide of the modulator. For example, the modulating groupcan be directly attached by covalent coupling to the β-amyloid peptideor the modulating group can be attached indirectly by a stablenon-covalent association. In one embodiment of the invention, themodulating group is attached to the amino-terminus of the β-amyloidpeptide of the modulator. Accordingly, the modulator can comprise acompound having a formula: ##STR3## Alternatively, in another embodimentof the invention, the modulating group is attached to thecarboxy-terminus of the β-amyloid peptide of the modulator. Accordingly,the modulator can comprise a compound having a formula: ##STR4## In yetanother embodiment, the modulating group is attached to the side chainof at least one amino acid residue of the β-amyloid peptide of thecompound (e.g., through the epsilon amino group of a lysyl residue(s),through the carboxyl group of an aspartic acid residue(s) or a glutamicacid residue(s), through a hydroxy group of a tyrosyl residue(s), aserine residue(s) or a threonine residue(s) or other suitable reactivegroup on an amino acid side chain).

The modulating group is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. Accordingly, since the β-AP peptide of thecompound is modified from its natural state, the modulating group "A" asused herein is not intended to include hydrogen. In a preferredembodiment, the modulating group is a biotin compound of the formula:##STR5## wherein X₁ -X₃ are each independently selected from the groupconsisting of S, O and NR₂ ¹ wherein R¹ is hydrogen, or an aryl, loweralkyl, alkenyl or alkynyl moiety; W is ═O or N(R¹)₂ R₁ is a loweralkylenyl moiety and Y is a direct bond or a spacer molecule selectedfor its ability to react with a target group on a β-AP. At least one ofX₁ -X₃ or W is an N(R¹)₂ group.

The term "aryl" is intended to include aromatic moieties containingsubstituted or unsubstituted ring(s), e.g., benzyl, naphthyl, etc. Othermore complex fused ring moieties also are intended to be included.

The term "lower alkyl or alkylenyl moiety" refers to a saturated,straight or branched chain (or combination thereof) hydrocarboncontaining 1 to about 6 carbon atoms, more preferably from 1 to 3 carbonatoms. The terms "lower alkenyl moiety" and "lower alkynyl moiety" referto unsaturated hydrocarbons containing 1 to about 6 carbon atoms, morepreferably 1 to 3 carbon atoms. Preferably, R¹ contains 1 to 3 carbonatoms. Preferably, R₁ contains 4 carbon atoms.

The spacer molecule (Y) can be, for example, a lower alkyl group or alinker peptide, and is preferably selected for its ability to link witha free amino group (e.g., the α-amino group at the amino-terminus of aβ-AP). Thus, in a preferred embodiment, the biotin compound modifies theamino-terminus of a β-amyloid peptide.

Additional suitable modulating groups may include other cyclic andheterocyclic compounds and other compounds having similar steric "bulk".Non-limiting examples of compounds which can be used to modify a β-APare shown schematically in FIG. 2, and include N-acetylneuraminic acid,cholic acid, trans-4-cotininecarboxylic acid,2-imino-1-imidazolidineacetic acid, (S)-(-)-indoline-2-carboxylic acid,(-)-menthoxyacetic acid, 2-norbomaneacetic acid,γ-oxo-5-acenaphthenebutyric acid, (-)-2-oxo-4-thiazolidinecarboxylicacid, tetrahydro-3-furoic acid, 2-iminobiotin-N-hydroxysuccinimideester, diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride,5-(and 6-)-carboxyfluorescein (succinimidyl ester), fluoresceinisothiocyanate, and acetic acid (or derivatives thereof).

In a modulator of the invention, a single modulating group may beattached to a β-amyloid peptide (e.g., n=1 in the formula shown above)or multiple modulating groups may be attached to the peptide. The numberof modulating groups is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. However, n preferably is an integer between1 and 60, more preferably between 1 and 30 and even more preferablybetween 1 and 10 or 1 and 5.

Modulators of the invention can be prepared by standard techniques knownin the art. The peptide component of a modulator composed, at least inpart of a peptide, can be synthesized using standard techniques such asthose described in Bodansky, M. Principles of Peptide Synthesis,Springer Verlag, Berlin (1993) and Grant, G. A (ed.). SyntheticPeptides: A User's Guide, W. H. Freeman and Company, New York (1992).Automated peptide synthesizers are commercially available (e.g.,Advanced ChemTech Model 396; Milligen/Biosearch 9600). Additionally, oneor more modulating groups can be attached to a β-amyloid peptide bystandard methods, for example using methods for reaction through anamino group (e.g., the alpha-amino group at the amino-terminus of apeptide or the epsilon amino group of a lysyl residue), a carboxyl group(e.g., at the carboxy terminus of a peptide or on an aspartic orglutamic acid residue), a hydroxyl group (e.g., on a tyrosine, serine orthreonine residue) or other suitable reactive group on an amino acidside chain (see e.g., Greene, T. W and Wuts, P. G. M. Protective Groupsin Organic Synthesis, John Wiley and Sons, Inc., New York (1991).Synthesis of preferred β amyloid modulators is described further inExample 1.

Another aspect of the invention pertains to a method for selecting amodulator of β-amyloid aggregation. In the method, a test compound iscontacted with natural β amyloid peptides, the aggregation of thenatural β-AP is measured and a modulator is selected based on theability of the test compound to alter the aggregation of the naturalβ-AP (e.g., inhibit or promote aggregation). In a preferred embodiment,the test compound is contacted with a molar excess amount of the naturalβ-AP. The amount and/or rate of natural β-AP aggregation in the presenceof the test compound can be determined by a suitable assay indicative ofβ-AP aggregation, as described herein.

In a preferred assay, the natural β-AP is dissolved in solution in thepresence of the test compound and aggregation of the natural β-AP isassessed by the turbidity of the solution over time, as determined bythe optical density of the solution (described further in Example 2; seealso Jarrett et al. (1993) Biochemistry 32:4693-4697). Typically, theabsorbance at 400 nm (A_(400nm)) of the β-AP solution is measured,either in the presence or absence of the test compound. In the absenceof a β-amyloid modulator, the A_(400nm) of the solution typically staysrelatively constant during a lag time in which the β-AP remains insolution but then the A_(400nm) of the solution rapidly increases as theβ-AP aggregates and comes out of solution, ultimately reaching a plateaulevel (i.e., the A_(400nm) of the solution exhibits sigmoidal kineticsover time). In contrast, in the presence of a test compound thatinhibits β-AP aggregation, the A_(400nm) of the solution is reducedcompared to when the modulator is absent. Thus, in the presence of theinhibitory modulator, the solution may exhibit an increased lag time, adecreased slope of aggregation and/or a lower plateau level compared towhen the modulator is absent. This method for selecting a modulator ofβ-amyloid polymerization can similarly be used to select modulators thatpromote β-AP aggregation. Thus, in the presence of a modulator thatpromotes β-AP aggregation, the A_(400nm) of the solution is increasedcompared to when the modulator is absent (e.g., the solution may exhibitan decreased lag time, increase slope of aggregation and/or a higherplateau level compared to when the modulator is absent).

Other assays suitable for use in the screening method of the inventionare described further in the Examples (e.g., Example 2 and Example 5).In one type of screening method, aggregation can be assayed based onenhanced emission of the dye Thioflavine T when contacted with β-AP orby visualization of high molecular weight β-AP aggregates on SDS-PAGEgels. Moreover, β-AP aggregation can be assessed by electron microscopy(EM) of the β-AP preparation in the presence or absence of themodulator. For example, β amyloid fibril formation, which is detectableby EM, is reduced in the presence of a modulator that inhibits β-APaggregation (i.e., there is a reduced amount or number of β-fibrils inthe presence of the modulator), whereas β fibril formation is increasedin the presence of a modulator that promotes β-AP aggregation (i.e.,there is an increased amount or number of β-fibrils in the presence ofthe modulator).

Another aspect of the invention pertains to pharmaceutical compositionsof the β-amyloid modulators of the invention. These compositions includea β amyloid modulator in a therapeutically effective amount sufficientto alter, and preferably inhibit, aggregation of natural β-amyloidpeptides, and a pharmaceutically acceptable carrier. A "therapeuticallyeffective amount" refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired result. Atherapeutically effective amount of modulator may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the modulator to elicit a desiredresponse in the individual. Dosage regimens may be adjusted to providethe optimum therapeutic response. A therapeutically effective amount isalso one in which any toxic or detrimental effects of the modulator areoutweighed by the therapeutically beneficial effects. The potentialneurotoxicity of the modulators of the invention can be assayed asdescribed in Example 3 and a therapeutically effective modulator can beselected which does not exhibit significant neurotoxicity. In apreferred embodiment, a therapeutically effective amount of a modulatoris sufficient to alter, and preferably inhibit, aggregation of a molarexcess amount of natural β-amyloid peptides.

One factor that may be considered when determining a therapeuticallyeffective amount of a β amyloid modulator is the concentration ofnatural β-AP in a biological compartment of a subject, such as in thecerebrospinal fluid (CSF) of the subject. The concentration of naturalβ-AP in the CSF has been estimated at 3 nM (Schwartzman, (1994) Proc.Natl. Acad. Sci. USA 91:8368-8372). A non-limiting range for atherapeutically effective amount of a β amyloid modulator is 0.01 nM-10μM. It is to be noted that dosage values may vary with the severity ofthe condition to be alleviated. It is to be further understood that forany particular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

As used herein "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forparenteral administration. More preferably, the carrier is suitable foradministration into the central nervous system (e.g., intraspinally orintracerebrally). Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin. Moreover, the modulators can beadministered in a time release formulation, for example in a compositionwhich includes a slow release polymer. The active compounds can beprepared with carriers that will protect the compound against rapidrelease, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., β-amyloid modulator) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

The amount of active compound in the composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual, each of which may affect the amount of natural β-AP in theindividual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

In another embodiment, a pharmaceutical composition of the invention isprovided as a packaged formulation. The packaged formulation may includea pharmaceutical composition of the invention in a container and printedinstructions for administration of the composition for treating asubject having a disorder associated with β-amyloidosis, e.g.Alzheimer's disease.

Another aspect of the invention pertains to methods for alteringaggregation of natural β-amyloid peptides. In the methods of theinvention, natural β amyloid peptides are contacted with a β amyloidmodulator such that aggregation of the natural β amyloid peptides isaltered. In one embodiment, the modulator inhibits aggregation of thenatural β amyloid peptides. In another embodiment, the modulatorpromotes aggregation of the natural β amyloid peptides. Preferably,aggregation of a molar excess amount of β-AP, relative to the amount ofmodulator, is altered upon contact with the modulator.

In the method of the invention, natural β amyloid peptides can becontacted with a modulator either in vitro or in vivo. Thus, the term"contacted with" is intended to encompass both incubation of a modulatorwith a natural β-AP preparation in vitro and delivery of the modulatorto a site in vivo where natural β-AP is present. Thus, in oneembodiment, the methods of the invention for altering β-AP aggregation,and the compounds utilized therein, are used in vitro, for example todetect and quantitate natural β-AP in sample (e.g., a sample ofbiological fluid). The source of natural β-AP used in the method can be,for example, a sample of cerebrospinal fluid (e.g., from an AD patient,an adult susceptible to AD due to family history, or a normal adult).The natural β-AP sample is contacted with a modulator of the inventionand aggregation of the β-AP is measured, such as by as assay describedin Example 2. The degree of aggregation of the β-AP sample can then becompared to that of a control sample(s) of a known concentration ofβ-AP, similarly contacted with the modulator and the results can be usedas an indication of whether a subject is suceptible to or has a disorderassociated with β-amyloidosis. Moreover, β-AP can be detected bydetecting a modulating group incorporated into the modulator. Forexample, modulators incorporating a biotin compound as described herein(e.g., an amino-terminally biotinylated β-AP peptide) can be detectedusing a streptavidin or avidin probe which is labeled with a detectablesubstance (e.g., an enzyme, such as peroxidase). Detection of naturalβ-AP aggregates mixed with a modulator of the invention using a probethat binds to the modulating group (e.g., biotin/streptavidin) isdescribed further in Example 2.

In a preferred embodiment, the method of the invention for alteringnatural β-AP aggregation is used therapeutically in the treatment ofdisorders associated with β amyloidosis, e.g., Alzheimer's Disease. Amodulator of the invention can be contacted with natural β amyloidpeptides present in a subject (e.g., in the cerebrospinal fluid orcerebrum of the subject) to thereby alter the aggregation of the naturalβ-AP. The modulator may be administered to a subject by any suitableroute effective for inhibiting natural β-AP aggregation in the subject,although in a particularly preferred embodiment, the modulator isadministered parenterally, most preferably to the central nervous systemof the subject. Possible routes of administration include intraspinaladministration and intracerebral administration (e.g.,intracerebrovascular administration).

Suitable modes and devices for delivery of therapeutic compounds to theCNS of a subject are known in the art, including cerebrovascularreservoirs (e.g., Ommaya or Rikker reservoirs; see e.g., Raney, J. P. etal. (1988) J. Neurosci. Nurs. 20:23-29, Sundaresan, N. et al. (1989)Oncology 3:15-22), catheters for intrathecal delivery (e.g.,Port-a-Cath, Y-catheters and the like; see e.g., Plummer, J. L. (1991)Pain 44:215-220; Yaksh, T. L. et al. (1986) Pharmacol. Biochem. Behav.25:483-485), injectable intrathecal reservoirs (e.g., Spinalgesic; seee.g., Brazenor, G. A. (1987) Neurosurgery 21:484-491), implantableinfusion pump systems (e.g., Infusaid; see e.g., Zierski, J. et al.(1988) Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am.Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by AlzaCorporation). A particularly preferred mode of administration is via animplantable, externally programmable infusion pump. Suitable infusionpump systems and reservoir systems are also described in U.S. Pat. No.5,368,562 by Blomquist and U.S. Pat. No. 4,731,058 by Doan, developed byPharmacia Deltec Inc.

The method of the invention for altering β-AP aggregation in vivo , andin particular for inhibiting β-AP aggregation, can be usedtherapeutically in diseases associated with abnormal β amyloidaggregation and deposition to slow the rate of β amyloid depositionand/or lessen the degree of β amyloid deposition, thereby amelioratingthe course of the disease. In a preferred embodiment, the method is usedto treat Alzheimer's disease (e.g., sporadic or familial AD, includingboth individuals exhibiting symptoms of AD and individuals susceptibleto familial AD). The method can also be used therapeutically to treatother clinical occurrences of β amyloid deposition, such as in Down'ssyndrome individuals and in patients with hereditary cerebral hemorrhagewith amyloidosis-Dutch-type (HCHWAD). While inhibition of β-APaggregation is a preferred therapeutic method, modulators that promoteβ-AP aggregation may also be useful therapeutically by allowing for thesequestration of β-AP at sites that do not lead to neurologicalimpairment.

This invention is further illustrated by the following examples whichshould not be construed as limiting. A modulator's ability to alter theaggregation of β-amyloid peptide in the assays described below arepredictive of the modulator's ability to perform the same function invivo. The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

EXAMPLE 1 Construction of β-Amyloid Modulators

A β-amyloid modulator composed of an amino-terminally biotinylatedβ-amyloid peptide of the amino acid sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

(positions 1 to 40 of SEQ ID NO: 1) was prepared by solid-phase peptidesynthesis using an N.sup.α -9-fluorenylmethyloxycarbonyl (FMOC)-basedprotection strategy as follows. Starting with 2.5 mmoles ofFMOC-Val-Wang resin, sequential additions of each amino acid wereperformed using a four-fold excess of protected amino acids,1-hydroxybenzotriazole (HOBt) and diisopropyl carbodiimide (DIC).Recouplings were performed when necessary as determined by ninhydrintesting of the resin after coupling. Each synthesis cycle was minimallydescribed by a three minute deprotection (25%piperidine/N-methyl-pyrrolidone (NMP)), a 15 minute deprotection, fiveone minute NMP washes, a 60 minute coupling cycle, five NMP washes and aninhydrin test. To a 700 mg portion of the fully assembledpeptide-resin, biotin (obtained commercially from Molecular Probes,Inc.) was substituted for an FMOC-amino acid was coupled by the aboveprotocol. The peptide was removed from the resin by treatment withtrifluoroacetic acid (TFA) (82.5%), water (5%), thioanisole (5%), phenol(5%), ethanedithiol (2.5%) for two hours followed by precipitation ofthe peptide in cold ether. The solid was pelleted by centrifugation(2400 rpm×10 min.), and the ether decanted. It was resuspended in ether,pelleted and decanted a second time. The solid was dissolved in 10%acetic acid and lyophilized to dryness to yield 230 mg of crudebiotinylated peptide. 60 mg of the solid was dissolved in 25%acetonitrile (ACN) /0.1% TFA and applied to a C18 reversed phase highperformance liquid chromatography (HPLC) column. Biotinyl βAP₁₋₄₀ waseluted using a linear gradient of 30-45% acetonitrile/0.1% TFA over 40minutes. One primary fraction (4 mg) and several side fractions wereisolated. The main fraction yielded a mass spectrum of 4556(matrix-assisted laser desorption ionization-time of flight) whichmatches the theoretical (4555) for this peptide.

A β-amyloid modulator composed of an amino-terminally biotinylatedβ-amyloid peptide of the amino acid sequence:

DAEFRHDSGYEVHHQ

(positions 1 to 15 of SEQ ID NO: 1) was prepared on an Advanced ChemTechModel 396 multiple peptide synthesizer using an automated protocolestablished by the manufacturer for 0.025 mmole scale synthesis. Doublecouplings were performed on all cycles using2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N,N-diisopropylethylamine (DIEA)/HOBt/FMOC-AA in four-fold excessfor 30 minutes followed by DIC/HOBt/FMOC-AA in four-fold excess for 45minutes. The peptide was deprotected and removed from the resin bytreatment with TFA/water (95%/5%) for three hours and precipitated withether as described above. The pellet was resuspended in 10% acetic acidand lyophilized. The material was purified by a preparative HPLC using15%-40% acetonitrile over 80 minutes on a Vydac C18 column (21×250 mm).The main isolate eluted as a single symmetrical peak when analyzed byanalytical HPLC and yielded the expected molecular weight when analyzedby electrospray mass spectrometry. Result=2052.6 (2052 theoretical).

EXAMPLE 2 Inhibition of β-Amyloid Aggregation by Modulators

The ability of β-amyloid modulators to inhibit the aggregation ofnatural β-AP when combined with the natural β-AP was examined in aseries of aggregation assays. Natural β-AP (β-AP₁₋₄₀) was obtainedcommercially from Bachem (Torrance, Calif.). Amino-terminallybiotinylated β-AP modulators were prepared as described in Example 1.

A. Optical Density Assay

In one assay, β-AP aggregation was measured by determining the increasein turbidity of a solution of natural β-AP over time in the absence orpresence of various concentrations of the modulator. Turbidity of thesolution was quantitated by determining the optical density at 400 nm(A_(400nm)) of the solution over time.

The aggregation of natural β-AP in the absence of modulator wasdetermined as follows. β-AP₁₋₄₀ was dissolved in hexafluoro isopropanol(HFIP; Aldrich Chemical Co., Inc.) at 2 mg/ml. Aliquots of the HFIPsolution (87 μl) were transferred to individual 10 mm×75 mm test tubes.A stream of argon gas was passed through each tube to evaporate theHFIP. To the resulting thin film of peptide, dimethylsulfoxide (DMSO;Aldrich Chemical Co., Inc.) (25 μl) was added to dissolve the peptide. A2 mm×7 mm TEFLON™-coated magnetic stir bar was added to each tube.Buffer (475 μL of 100 mM NaCl, 10 mM sodium phosphate, pH 7.4) was addedto the DMSO solution with stirring. The resulting mixture was stirredcontinuously and the optical density was monitored at 400 nm to observethe formation of insoluble peptide aggregates.

Alternatively, β-AP₁₋₄₀ was dissolved in DMSO as described above at 1.6mM (6.9 mg/ml) and aliquots (25 μl) were added to stirred buffer (475μl), followed by monitoring of absorbance at 400 nm.

For inhibition studies in which a β-amyloid modulator was dissolved insolution together with the natural β-AP, the modulators were dissolvedin DMSO either with or without prior dissolution in HFIP. Thesecompounds were then added to buffer with stirring, followed by additionof β-AP₁₋₄₀ in DMSO. Alternatively, HFIP solutions of modulators werecombined with β-AP₁₋₄₀ in HFIP followed by evaporation and redissolutionof the mixture in DMSO. Buffer was then added to the DMSO solution toinitiate the assay. The amino-terminally biotinylated β-amyloid peptidemodulators N-biotinyl-βAP₁₋₄₀, and N-biotinyl-βAP₁₋₁₅ were tested atconcentrations of 1% and 5% in the natural β-AP₁₋₄₀ solution.

A representative example of the results is shown graphically in FIG. 1,which depicts the inhibition of aggregation of natural β-AP₁₋₄₀ byN-biotinyl-βAP₁₋₄₀. In the absence of the modulator, the optical densityof the natural β-AP solution showed a characteristic sigmoidal curve,with a lag time prior to aggregation (approximately 3 hours in FIG. 1)in which the A_(400nm) was low, followed by rapid increase in theA_(400nm), which quickly reached a plateau level, representingaggregation of the natural β amyloid peptides. In contrast, in thepresence of as little as 1% of the N-biotinyl-βAP₁₋₄₀ modulator,aggregation of the natural β amyloid peptides was markedly inhibited,indicated by an increase in the lag time, a decrease in the slope ofaggregation and a decrease in the plateau level reached for theturbidity of the solution (see FIG. 1). N-biotinyl-βAP₁₋₄₀ at aconcentration of 5% similarly inhibited aggregation of the natural βamyloid peptide. Furthermore, similar results were observed whenN-biotinyl-βAP₁₋₁₅ was used as the modulator. These results demonstratethat an N-terminally biotinylated β-AP modulator can effectively inhibitthe aggregation of natural β amyloid peptides, even when the natural βamyloid peptides are present at as much as a 100-fold molar excessconcentration.

B. Fluorescence Assay

In a second assay, β-AP aggregation was measured using a fluorometricassay essentially as described in Levine, H. (1993) Protein Science2:404-410. In this assay, the dye thioflavine T (ThT) is contacted withthe β-AP solution. Association of ThT with aggregated β-AP, but notmonomeric or loosely associated β-AP, gives rise to a new excitation(ex) maximum at 450 nm and an enhanced emission (em) at 482 nm, comparedto the 385 nm (ex) and 445 nm (em) for the free dye. β-AP aggregationwas assayed by this method as follows. Aliquots (2.9 μl) of thesolutions used in the aggregation assays as described above in section Awere removed from the samples and diluted in 200 μl of potassiumphosphate buffer (50 mM, pH 7.0) containing thioflavin T (10 μM;obtained commercially from Aldrich Chemical Co., Inc.). Excitation wasset at 450 nm and emission was measured at 482 nm. Similar to theresults observed with the optical density assay described above insection A, as little as 1% of the N-biotinylated β-AP modulators waseffective at inhibiting the aggregation of natural β amyloid peptidesusing this fluorometric assay.

C. Static Aggregation Assay

In a third assay, β-AP aggregation was measured by visualization of thepeptide aggregates using SDS-polyacrylamide gel electrophoresis(SDS-PAGE). In this assay, β-AP solutions were allowed to aggregate overa period of time and then aliquots of the reaction were run on astandard SDS-PAGE gel. Typical solution conditions were 200 μM ofβ-AP₁₋₄₀ in PBS at 37° C. for 8 days or 200 μM β-AP₁₋₄₀ in 0.1M sodiumacetate at 37° C. for 3 days. The peptide aggregates were visualized byCoomassie blue staining of the gel or, for β-AP solutions that includeda biotinylated β-AP modulator, by western blotting of a filter preparedfrom the gel with a streptavidin-peroxidase probe, followed by astandard peroxidase assay. The β-AP aggregates are identifiable as highmolecular weight, low mobility bands on the gel, which are readilydistinguishable from the low molecular weight, high mobility β-APmonomer or dimer bands.

When natural β-AP₁₋₄₀ aggregation was assayed by this method in theabsence of any β amyloid modulators, high molecular weight aggregateswere readily detectable on the gel. In contrast, whenN-biotinyl-β-AP₁₋₄₀ modulator self-aggregation was assayed (i.e.,aggregation of the N-biotinyl peptide alone, in the absence of anynatural β-AP), few if any high molecular weight aggregates wereobserved, indicating that the ability of the modulator to self-aggregateis significantly reduced compared to natural β-AP. Finally, whenaggregation of a mixture of natural β-AP₁₋₄₀ and N-biotinylated β-AP₁₋₄₀was assayed by this method, reduced amounts of the peptide mixtureassociated into high molecular weight aggregates, thus demonstratingthat the β amyloid modulator is effective at inhibiting the aggregationof the natural β amyloid peptides.

EXAMPLE 3 Neurotoxicity Analysis of β-Amyloid Modulators

The neurotoxicity of the β-amyloid modulators is tested in a cell-basedassay using the neuronal precursor cell line PC-12, or primary neuronalcells, and the viability indicator3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). (SeeShearman, M. S. et al. (1994) Proc. Natl. Acad. Sci. USA 91:1470-1474;Hansen, M. B. et al. (1989) J. Immun. Methods 119:203-210). PC-12 is arat adrenal pheochromocytoma cell line and is available from theAmerican Type Culture Collection, Rockville, Md. (ATCC CRL 1721). MTT(commercially available from Sigma Chemical Co. ) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

To test the neurotoxicity of a β-amyloid modulator (either alone orcombined with natural β-AP), cells first are plated in 96-well plates at7,000-10,000 cells/well and allowed to adhere by overnight culture at37° C. Serial dilutions of freshly dissolved or "aged" modulators(either alone or combined with natural β-AP) in phosphate bufferedsaline (PBS) are added to the wells in triplicate and incubation iscontinued for two or more days. Aged modulators are prepared byincubating an aqueous solution of the modulator at 37° C. undisturbedfor a prolonged period (e.g., five days or more). For the final twohours of exposure of the cells to the modulator preparation, MTT isadded to the media to a final concentration of 1 mg/ml and incubation iscontinued at 37° C. Following the two hour incubation with MTT, themedia is removed and the cells are lysed in isopropanol/0.4N HCl withagitation. An equal volume of PBS is added to each well and theabsorbance of each well at 570 nm is measured to quantitate viablecells. Alternatively, MTT is solubilized by addition of 50% N,N-dimethylformamide/20% sodium dodecyl sulfate added directly to the media in thewells and viable cells are likewise quantitated by measuring absorbanceat 570 nm. The relative neurotoxicity of a β-amyloid modulator (eitheralone or in combination with natural β-AP is determined by comparison tonatural β-AP alone (e.g., β1-40, β1-42), which exhibits neurotoxicity inthis assay and thus can serve as a positive control.

EXAMPLE 4 Syntheses of Additional Modified β-Amyloid Peptide Compounds

In this example, a series of modified β-APs, having a variety ofN-terminal or random side chain modifications were sythesized.

A series of N-terminally modified β-amyloid peptides was synthesizedusing standard methods. Fully-protected resin-bound peptidescorresponding to Aβ(1-15) and Aβ(1-40) were prepared as described inExample 1 on Wang resin to eventually afford carboxyl terminal peptideacids. Small portions of each peptide resin (13 and 20 μmoles,respectively) were aliquoted into the wells of the reaction block of anAdvanced ChemTech Model 396 Multiple Peptide Synthesizer. The N-terminalFMOC protecting group of each sample was removed in the standard mannerwith 25% piperidine in NMP followed by extensive washing with NMP. Theunprotected N-terminal a-amino group of each peptide-resin sample wasmodified using one of the following methods:

Method A, coupling of modifying reagents containing free carboxylic acidgroups: The modifying reagent (five equivalents) was predissolved inNMP, DMSO or a mixture of these two solvents. HOBT and DIC (fiveequivalents of each reagent) were added to the dissolved modifier andthe resulting solution was added to one equivalent of free-aminopeptide-resin. Coupling was allowed to proceed overnight, followed bywashing. If a ninhydrin test on a small sample of peptide-resin showedthat coupling was not complete, the coupling was repeated using1-hydroxy-7-azabenzotriazole (HOAt) in place of HOBt.

Method B, coupling of modifying reagents obtained in preactivated forms:The modifying reagent (five equivalents) was predissolved in NMP, DMSOor a mixture of these two solvents and added to one equivalent ofpeptide-resin. Diisopropylethylamine (DIEA; six equivalents) was addedto the suspension of activated modifier and peptide-resin. Coupling wasallowed to proceed overnight, followed by washing. If a ninhydrin teston a small sample of peptide-resin showed that coupling was notcomplete, the coupling was repeated.

After the second coupling (if required) the N-terminally modifiedpeptide-resins were dried at reduced pressure and cleaved from the resinwith removal of side-chain protecting groups as described in Example 1.Analytical reversed-phase HPLC was used to confirm that a major productwas present in the resulting crude peptides which were purified usingMillipore Sep-Pak cartridges or preparative reverse-phase HPLC. Massspectrometry was used to confirm the presence of the desired compound inthe product.

Method A was used to couple N-acetylneuraminic acid, cholic acid,trans-4-cotininecarboxylic acid, 2-imino-1-imidazolidineacetic acid,(S)-(-)-indoline-2-carboxylic acid, (-)-menthoxyacetic acid,2-norbornaneacetic acid, γ-oxo-5-acenaphthenebutyric acid,(-)-2-oxo-4-thiazolidinecarboxylic acid, and tetrahydro-3-furoic acid.Method B was used to couple 2-iminobiotin-N-hydroxysuccinimide ester,diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, and 2-thiophenesulfonyl chloride.

In a manner similar to the construction of N-terminally modifiedAβ(1-15) and Aβ(1-40) peptides described above, N-fluoresceinyl Aβ(1-15)and Aβ(1-40) were prepared in two alternative manners using thepreactivated reagents 5-(and 6)-carboxyfluorescein succinimidyl esterand fluorescein-5-isothiocyanate (FITC Isomer I). Both reagents wereobtained from Molecular Probes Inc. Couplings were performed using fourequivalents of reagent per equivalent of pepitde-resin with DIEA addedto make the reaction solution basic to wet pH paper. Couplings of eachreagent to Aβ(1-15)-resin appeared to be complete after a singleovernight coupling. Coupling to Aβ(1-40)-resin was slower as indicatedby a positive ninhydrin test and both reagents were recoupled to thispeptide-resin overnight in tetrahydrofuran-NMP (1:2 v/v). The resultingN-terminally modified peptide-resins were cleaved, deprotected andpurified as described in Example A.

In addition to the N-fluoresceinyl Aβ peptides described above, aβ-amyloid modulator comprised of random modification of Aβ(1-40) withfluorescein was prepared. Aβ(1-40) purchased from Bachem was dissolvedin DMSO at approximately 2 mg/mL. 5-(and-6)-Carboxyfluorescein purchasedfrom Molecular Probes was added in a 1.5 molar excess and DIEA was addedto make the solution basic to wet pH paper. The reaction was allowed toproceed for 1 hour at room temperature and was then quenched withtriethanolamine. The product was added to assays as this crude mixture.

EXAMPLE 5 Identification of Additional β-Amyloid Modulators

In this Example, two assays of Aβ aggregation were used to identifyβ-amyloid modulators which can inhibit this process.

The first assay is referred to as a seeded static assay (SSA) and wasperformed as follows:

To prepare a solution of Aβ monomer, the appropriate quantity ofAβ(1-40) peptide (Bachem) was weighed out on a micro-balance (the amountwas corrected for the amount of water in the preparation, which,depending on lot number, was 20-30% w/w). The peptide was dissolved in1/25 volume of dimethysulfoxide (DMSO), followed by water to 1/2 volumeand 1/2 volume 2× PBS (10× PBS: NaCl 137 mM, KCl 2.7 mM Na₂ HPO₄ •7H₂ O4.3 mM, KH₂ PO₄ 1.4 mM pH 7.2) to a final concentration of 200 μM.

To prepare a stock seed, 1 ml of the above Aβ monomer preparation, wasincubated for 8 days at 37° C. and sheared sequentially through an 18,23, 26 and 30 gauge needle 25, 25, 50, and 100 times respectively. 2 μlsamples of the sheared material was taken for fluorescence measurementsafter every 50 passes through the 30 gauge needle until the fluorescenceunits (FU) had plateaued (approx. 100-150×).

To prepare a candidate inhibitor, the required amount of candidateinhibitor was weighed out and the stock dissolved in 1× PBS to a finalconcentration of 1 mM (10× stock). If insoluble, it was dissolved in1/10 volume of DMSO and diluted in 1× PBS to 1 mM. A further 1/10dilution was also prepared to test each candidate at both 100 μM and 10μM.

For the aggregation assay, each sample was set up in triplicate 50 μl of200 μM monomer, 125 FU sheared seed (variable quantity dependent on thebatch of seed, routinely 3-6 μl), 10 μl of 10× inhibitor solution, finalvolume made up to 100 μl with 1× PBS!. Two concentrations of eachinhibitor were tested 100 μM and 10 μM, equivalent to a 1:1 and a 1:10molar ratio of monomer to inhibitor. The controls included an unseededreaction to confirm that the fresh monomer contained no seed, and aseeded reaction in the absence of inhibitor, as a reference to compareagainst putative inhibitors. The assay was incubated at 37° C. for 6 h,taking 2 μl samples hourly for fluorescence measurements. To measurefluorescence, a 2 μl sample of Aβ was added to 400 μl of Thioflavin-Tsolution (50 mM Potassium Phosphate 10 mM Thioflavin-T pH 7.5). Thesamples were vortexed and the fluorescence was read in a 0.5 ml microquartz cuvette at EX 450 nm and EM 482 nm (Hitachi 4500 Fluorimeter).β-aggregation results in enhanced emission of Thioflavin-T. Accordingly,samples including an effective inhibitor compound exhibit reducedemission as compared to control samples without the inhibitor compound.

The second assay is referred to as a shaken plate aggregation assay andwas performed as follows:

Aβ(1-40) peptide from Bachem (Torrance, Calif.) was dissolved in HFIP(1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8) at a concentrationof 2 mg peptide/ml and incubated at room temperature for 30 min. HFIPsolubilized peptide was sonicated in a waterbath sonicator for 5 min athighest setting, then evaporated to dryness under a stream of argon. Thepeptide film was resuspended in anhydrous dimethylsulfoxide (DMSO) at aconcentration of 6.9 mg/ml, sonicated for 5 min as before, then filteredthrough a 0.2 micron nylon syringe filter (VWR cat. No. 28196-050).Candidate inhibitors were dissolved directly in DMSO, generally at amolar concentration 4 times that of the Aβ(1-40) peptide.

Candidates were assayed in triplicate. For each candidate to be tested,4 parts Aβ(1-40) peptide in DMSO were combined with 1 part candidateinhibitor in DMSO in a glass vial, and mixed to produce a 1:1 molarratio of Aβ peptide to candidate. For different molar ratios, candidateswere diluted with DMSO prior to addition to Aβ(1-40), in order to keepthe final DMSO and Aβ(1-40) concentrations constant. Into an ultra lowbinding 96 well plate (Corning Costar cat. No. 2500, Cambridge, Mass.)100 μl PTL buffer (150 mM NaCl, 10 mM NaH₂ PO_(4;) pH 7.4) wasaliquotted per well. For each candidate, 10 μl of peptide mixture inDMSO was aliquotted into each of three wells containing buffer. Thecovered plate was vigorously vortexed on a plate shaker at high speedfor 30 seconds. An additional 100 μl of PTL buffer was added to eachwell and again the plate was vortexed vigorously for 30 sec. Absorbanceat 405 nm was immediately read in a plate reader for a baseline reading.The plate was returned to the plate shaker and vortexed at moderatespeed for 5 hours at room temperature, with absorbance readings taken at15-20 min intervals. Increased absorbance indicated aggregation.Accordingly, effective inhibitor compounds cause a decrease inabsorbance in the test sample as compared to a control sample withoutthe inhibitor compound.

Representative results of the static seeded assay and shaken plate assaywith preferred β-amyloid modulators are shown below in Table I.

                                      TABLE I                                     __________________________________________________________________________                                       Effect in                                                                             Effect in                          Candidate                                                                          Aβ Amino                                                                      Modifying                shaken plate                                                                          Seeded Static                      Inhibitor                                                                          Acids                                                                              Reagent                  assay   Assay*                             __________________________________________________________________________    174  Aβ1-15                                                                        Cholic acid              Complete                                                                              ++                                                                    inhibition at                                                                 100% conc                                  176  Aβ1-15                                                                        Diethylene-              Decreased                                                                             ++                                           triamine penta           Plateau                                              acetic acid                                                         180  Aβ1-15                                                                        (-)-Menthoxy             None    ++                                           acetic acid                                                         190  Aβ1-15                                                                        Fluorescein              Decreased                                                                             ++                                           carboxylic acid          Plateau                                              (FICO)                                                              220  Aβ16-40                                                                       NH.sub.2 -EVHHHHQQK- Aβ(16-40)!-COOH(SEQ ID NO:                                                   Complete                                                                              ++                                      mutant                        inhibition at                                                                 100%, increased                                                               lag at 10%                                 224  Aβ1-40                                                                        F.sub.19 F.sub.20 ->T.sub.19 T.sub.20                                                                  Increased lag                                                                         ++                                      mutant                                                                   233  A6β-20                                                                        Acetic acid              accelerated                                                                           ++                                                                    aggregation at                                                                10% conc                                   __________________________________________________________________________     *++ = A strong inhibitor of aggregation. The rate of aggregation in the       presence of the inhibitor was decreased compared to the control by at         least 30-50%                                                             

These results indicate that β-APs modified by a wide variety ofN-terminal modifying groups are effective at modulating β-amyloidaggregation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspAlaGluPheArgHisAspSerGlyTyrGluValHisHisGlnLys                              151015                                                                        LeuValPhePheAlaGluAspValGlySerAsnLysGlyAlaIleIle                              202530                                                                        GlyLeuMetValGlyGlyValValIleAlaThr                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluValLysMetAspAlaGluPheArgHisAspSerGlyTyrGluVal                              151015                                                                        HisHisGlnLysLeuValPhePheAlaGluAspValGlySerAsnLys                              202530                                                                        GlyAlaIleIleGlyLeuMetValGlyGlyValValIleAlaThrVal                              354045                                                                        IleValIleThrLeuValMetLeuLysLysLysGlnTyrThrSerIle                              505560                                                                        HisHisGlyValValGluValAspAlaAlaValThrProGluGluArg                              65707580                                                                      HisLeuSerLysMetGlnGlnAsnGlyTyrGluAsnProThrTyrLys                              859095                                                                        PhePheGluGlnMetGlnAsn                                                         100                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified site                                                   (B) LOCATION: 19, 20                                                          (D) OTHER INFORMATION: /note= "Xaa"is a hydrophobic amino                     acid."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AspAlaGluPheArgHisAspSerGlyTyrGluValHisHisGlnLys                              151015                                                                        LeuValXaaXaaAlaGluAspValGlySerAsnLysGlyAlaIleIle                              202530                                                                        GlyLeuMetValGlyGlyValValIleAlaThr                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GluValHisHisHisHisGlnGlnLysLysLeuValPhePheAlaGlu                              151015                                                                        AspValGlySerAsnLysGlyAlaIleIleGlyLeuMetValGlyGly                              202530                                                                        ValVal                                                                        __________________________________________________________________________

We claim:
 1. A method for inhibiting aggregation of natural β-amyloidpeptides, comprising contacting the natural β-amyloid peptides with acompound such that aggregation of the natural β-amyloid peptides isinhibited, said compound having a formula: ##STR6## wherein Xaa is aβ-amyloid peptide, A is a cyclic or heterocyclic modulating groupattached to the β-amyloid peptide of the compound such that the compoundinhibits aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides, and n is an integer selected such thatthe compound inhibits aggregation of natural β-amyloid peptides whencontacted with the natural β-amyloid peptides.
 2. The method of claim 1,wherein at least one A group is attached to the amino terminus of theβ-amyloid peptide of the compound.
 3. The method of claim 2, wherein theat least one A group comprises a biotin compound of the formula:##STR7## wherein X₁ -X₃ are each independently selected from the groupconsisting of S, O and NR', wherein R' is selected from the groupconsisting of hydrogen, an aryl moiety, a lower alkyl moiety, an alkenylmoiety and an alkynyl moiety;W is ═O or N(R')₂ ; R₁ is a lower alkylenylmoiety; and Y is a direct bond or a spacer molecule selected for itsability to react with an amino group, whereby at least one of X₁ -X₃ isan NR' group or W is an N(R')₂ group.
 4. The method of claim 2, whereinthe at least one A group is attached to the β-amyloid peptide bymodifying the β-amyloid peptide with a compound selected from the groupconsisting of diethylenetriaminepentaacetic dianhydride, cholic acid,(-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluoresceinisothiocyanate and acetic acid.
 5. The method of claim 1, wherein thenatural β-amyloid peptides are present in a subject and the compound isadministered to the central nervous system of the subject.
 6. A methodfor treating a subject for a disorder associated with β-amyloidosis,comprising:administering to the subject a therapeutically effectiveamount of a modulator of β-amyloid aggregation such that the subject istreated for a disorder associated with β-amyloidosis, said modulatorhaving a formula: ##STR8## wherein Xaa is a β-amyloid peptide, A is amodulating group attached to the β-amyloid peptide of the modulator suchthat the modulator inhibits aggregation of natural β-amyloid peptideswhen contacted with the natural β-amyloid peptides, and n is an integerselected such that the modulator inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.7. The method of claim 6, wherein the disorder is Alzheimer's disease.8. A method for treating a subject for a disorder associated withβ-amyloidosis, comprising:administering to the subject a therapeuticallyeffective amount of a compound comprising a retro-inverso isomer of aβ-amyloid peptide, wherein said compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides,such that the subject is treated for a disorder associated withβ-amyloidosis.
 9. The method of claim 8, wherein the disorder isAlzheimer's disease.
 10. The method of claim 6, wherein the modulatinggroup comprises a cyclic or heterocyclic compound.
 11. The method ofclaim 8, wherein the retro-inverso isomer of the β-amyloid peptidefurther comprises a modulating group attached to the amino-terminal endof the retro-inverso isomer.
 12. The method of claim 11, wherein themodulating group comprises a cyclic or heterocyclic compound.
 13. Themethod of claim 8, wherein the retro-inverso isomer of the β-amyloidpeptide further comprises a modulating group attached to thecarboxy-terminal end of the retro-inverso isomer.
 14. The method ofclaim 13, wherein the modulating group comprises a cyclic orheterocyclic compound.
 15. The method of claim 8, wherein theretro-inverso isomer of the β-amyloid peptide further comprisesmodulating groups attached to the amino- and carboxy-terminal ends ofthe retro-inverso isomer.
 16. A method for inhibiting aggregation ofnatural β-amyloid peptides, comprising contacting the natural β-amyloidpeptides with a compound comprising a retro-inverso isomer of aβ-amyloid peptide such that aggregation of the natural β-amyloidpeptides is inhibited.
 17. The method of claim 16, wherein theretro-inverso isomer of the β-amyloid peptide further comprises amodulating group attached to the amino-terminal end of the retro-inversoisomer.
 18. The method of claim 17, wherein the modulating groupcomprises a cyclic or heterocyclic compound.
 19. The method of claim 16,wherein the retro-inverso isomer of the β-amyloid peptide furthercomprises a modulating group attached to the carboxy-terminal end of theretro-inverso isomer.
 20. The method of claim 19, wherein the modulatinggroup comprises a cyclic or heterocyclic compound.
 21. The method ofclaim 16, wherein the retro-inverso isomer of the β-amyloid peptidefurther comprises modulating groups attached to the amino- andcarboxy-terminal ends of the retro-inverso isomer.
 22. The method ofclaim 16, wherein the natural β-amyloid peptides are present in asubject and the compound is administered to the central nervous systemof the subject.
 23. A method for inhibiting aggregation of naturalβ-amyloid peptides, comprising contacting the natural β-amyloid peptideswith a compound such that aggregation of the natural β-amyloid peptidesis inhibited, said compound having a formula: ##STR9## wherein Xaa is aβ-amyloid peptide and A is a modulating group attached to theamino-terminus of the β-amyloid peptide such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides.
 24. The method of claim 23, wherein themodulating group comprises a biotin compound of the formula: ##STR10##wherein X₁ -X₃ are each independently selected from the group consistingof S, O and NR', wherein R' is selected from the group consisting ofhydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and analkynyl moiety;W is ═O or N(R')₂ ; R₁ is a lower alkylenyl moiety; and Yis a direct bond or a spacer molecule selected for its ability to reactwith an amino group, whereby at least one of X₁ -X₃ is an NR' group or Wis an N(R')₂ group.
 25. The method of claim 23, wherein the modulatinggroup is attached to the β-amyloid peptide by modifying the β-amyloidpeptide with a compound selected from the group consisting ofdiethylenetriaminepentaacetic dianhydride, cholic acid,(-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluoresceinisothiocyanate and acetic acid.
 26. The method of claim 23, wherein themodulating group comprises a cyclic or heterocyclic compound.
 27. Themethod of claim 23, wherein the natural β-amyloid peptides are presentin a subject and the compound is administered to the central nervoussystem of the subject.
 28. A method for treating a subject for adisorder associated with β-amyloidosis, comprising:administering to thesubject a therapeutically effective amount of a modulator of β-amyloidaggregation such that the subject is treated for a disorder associatedwith β-amyloidosis, said modulator having a formula: ##STR11## whereinXaa is a β-amyloid peptide and A is a modulating group attached to theamino-terminus of the β-amyloid peptide such that the modulator inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides, such that the subject is treated for adisorder associated with β-amyloidosis.
 29. The method of claim 28,wherein the modulating group comprises a cyclic or heterocycliccompound.
 30. The method of claim 28, wherein the disorder isAlzheimer's disease.
 31. A method for inhibiting aggregation of naturalβ-amyloid peptides, comprising contacting the natural β-amyloid peptideswith a compound such that aggregation of the natural β-amyloid peptidesis inhibited, said compound having a formula: ##STR12## wherein Xaa is aβ-amyloid peptide and A is a modulating group attached to thecarboxy-terminus of the β-amyloid peptide such that the compoundinhibits aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides.
 32. The method of claim 31, wherein themodulating group comprises a biotin compound of the formula: ##STR13##wherein X₁ -X₃ are each independently selected from the group consistingof S, O and NR', wherein R' is selected from the group consisting ofhydrogen, an aryl moiety, a lower alkyl moiety, an alkenyl moiety and analkynyl moiety;W is ═O or N(R')₂ ; R₁ is a lower alkylenyl moiety; and Yis a direct bond or a spacer molecule selected for its ability to reactwith an amino group, whereby at least one of X₁ -X₃ is an NR' group or Wis an N(R')₂ group.
 33. The method of claim 31, wherein the modulatinggroup is attached to the β-amyloid peptide by modifying the β-amyloidpeptide with a compound selected from the group consisting ofdiethylenetriaminepentaacetic dianhydride, cholic acid,(-)-menthoxyacetic acid, 5-(and 6-)-carboxyfluorescein, fluoresceinisothiocyanate and acetic acid.
 34. The method of claim 31, wherein themodulating group comprises a cyclic or heterocyclic compound.
 35. Themethod of claim 31, wherein the natural β-amyloid peptides are presentin a subject and the compound is administered to the central nervoussystem of the subject.
 36. A method for treating a subject for adisorder associated with β-amyloidosis, comprising:administering to thesubject a therapeutically effective amount of a modulator of β-amyloidaggregation such that the subject is treated for a disorder associatedwith β-amyloidosis, said modulator having a formula: ##STR14## whereinXaa is a β-amyloid peptide and A is a modulating group attached to thecarboxy-terminus of the β-amyloid peptide such that the modulatorinhibits aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides, such that the subject is treated for adisorder associated with β-amyloidosis.
 37. The method of claim 36,wherein the modulating group comprises a cyclic or heterocycliccompound.
 38. The method of claim 36, wherein the disorder isAlzheimer's disease.